CN111995758B - Rare earth coordination polymer probe for detecting guanylic acid and preparation method and application thereof - Google Patents
Rare earth coordination polymer probe for detecting guanylic acid and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a rare earth coordination polymer probe for detecting guanylic acid. The prepared rare earth coordination polymer probe can be well dispersed in a water phase buffer solution and is more suitable for detecting biological samples; the rare earth coordination polymer probe is prepared by self-assembly reaction at room temperature, high temperature and longer reaction time are not needed, and the preparation process is simpler; the prepared rare earth coordination polymer probe has stable optical property, strong identifying capability on guanylic acid, quick response, response range of 0.15-20 mu M and detection limit of 0.10 mu M.
Description
Technical Field
The invention belongs to the technical field of fluorescent materials, and particularly relates to a rare earth coordination polymer probe for detecting guanylic acid, and a preparation method and application thereof.
Background
At present, guanosine-5' -monoposphate (GMP) is commonly used as an important additive for foods such as meat, chicken essence, and beverages in order to enhance the flavor and taste of foods. Furthermore, guanylic acid is also used in infant formula as a nutritional ingredient. However, any excessive intake of additives or nutrients can cause serious harm to the human body. Therefore, many countries have established strict limits for food additives or nutritional ingredients. According to the recommendations of the european union committee on the industrial dietario association (IDEACE), the maximum allowable amount of guanylic acid for low weight infant formulas was found to be 6.5 mg/100 kcal (326.3mg/kg) (j.nutr.,2002,132,1395S-1577S.); it is also well established in the Australian New food code that guanylic acid should be added to Infant Formula in amounts not higher than 3.8 mg/100 kg (797.69mg/kg) (Standard 2.9.1, Infant Formula products, Department of health and administration, 2000). According to the recommendations of the world food and agriculture organization and the world health organization, the maximum allowable addition amount of guanylic acid in meat products (luncheon meat, ham and bacon) is 0.5g/kg (500 mg/kg). Therefore, the establishment of the method for detecting the addition amount of guanylic acid in food is of great significance. At present, for the detection of guanylic acid, chromatographic methods (J.Chromatogr.A,2010,1217,5501-5510), electrochemical methods (Electrochimica Acta,2013.95,246-250; J.Anal.chem.,2015,70,186-192) and fluorescence analysis methods (J.Lumin.,2016,169, 173-181; Tetrahedron Lett.,2014,55, 6131-6136) have been developed, the chromatographic methods usually need a complicated solid phase extraction procedure, the electrochemical analysis chemical methods are difficult to avoid the interference of other substances and have low selectivity, the developed fluorescence methods usually need some complex organic synthesis, and the prepared probes have poor water solubility and are difficult to be used for the detection of guanylic acid in food.
Disclosure of Invention
The invention provides a rare earth coordination polymer probe for detecting guanylic acid and a preparation method and application thereof.
The object of the invention is achieved in the following way:
a method for preparing a rare earth coordination polymer probe for detecting guanylic acid comprises the steps of mixing a citrate solution and a rare earth metal salt solution, stirring at room temperature, performing full reaction, centrifuging, collecting white precipitate, and drying to obtain the rare earth coordination polymer probe for detecting guanylic acid.
Stirring and reacting for 2-6h at room temperature, centrifuging at 6000-8000 rpm, washing the collected white precipitate with deionized water, and drying at 60-80 deg.C.
The mole ratio of the citrate to the rare earth metal salt is (0.5-5): 5.
the rare earth metal ion in the rare earth metal salt solution is Tb3+、Eu3+At least one of (1).
The citrate is at least one of sodium citrate and potassium citrate.
The rare earth coordination polymer probe is prepared by the preparation method of the rare earth coordination polymer probe for detecting guanylic acid.
The method for detecting guanylic acid by utilizing the rare earth coordination polymer probe comprises the following specific steps: dissolving the rare earth coordination polymer probe in a water phase buffer solution to prepare a rare earth coordination polymer probe solution, adding the rare earth coordination polymer probe solution into the liquid to be detected, reacting for a certain time, detecting the fluorescence spectrum of the probe, and substituting the measured fluorescence intensity into a standard curve to obtain the concentration of guanylic acid in the liquid to be detected.
The standard curve is established as follows:
a: establishment of a standard curve for the detection of guanylic acid in aqueous solution: preparing guanylic acid standard solutions, adding guanylic acid standard solutions with different volumes into 10 mu L of rare earth coordination polymer probe suspension to enable the concentration of guanylic acid in the suspension to be changed in a gradient manner, fixing the total volume to be 100 mu L by adjusting the amount of HEPES buffer solution, reacting for 15 minutes at room temperature, and measuring the corresponding fluorescence intensity of the guanylic acid standard solutions at the excitation wavelength of 252 nm; establishing a standard curve by taking the fluorescence intensity as a vertical coordinate y and the concentration of guanylic acid in the suspension as a horizontal coordinate x, and drawing up a linear equation;
b: establishment of a standard curve for detecting guanylic acid in milk powder: preparing a guanylic acid standard solution, dissolving milk powder in water to obtain a milk powder aqueous solution, removing proteins in the milk powder by using an ultrafiltration centrifugal tube, adding different volumes of guanylic acid standard solutions into the milk powder aqueous solution to obtain milk powder aqueous solutions containing guanylic acid with different concentrations, adding 5 mu L of the milk powder aqueous solutions containing guanylic acid with different concentrations and 85 mu L of HEPES buffer solution into 10 mu L of rare earth coordination polymer probe suspension, fixing the total volume to 100 mu L, carrying out ultrasonic treatment for 20 minutes, and detecting the fluorescence intensity of the solution at 252nm excitation wavelength at room temperature; establishing a standard curve by taking the fluorescence intensity as a vertical coordinate y and taking the concentration of guanylic acid in the milk powder aqueous solution as a horizontal coordinate x, and drawing up a linear equation;
c: establishment of a standard curve for detection of guanylic acid in ham: preparing a guanylic acid standard solution, adding 4g of crushed ham into 2mL of deionized water, carrying out ultrasonic treatment for 1 hour, and centrifuging to remove impurities to obtain a ham aqueous solution; adding different volumes of guanylic acid standard solutions into the ham aqueous solution to obtain ham aqueous solutions containing guanylic acid with different concentrations; adding 5 mu L of ham aqueous solution with different concentrations of guanylic acid and 85 mu L of HEPES buffer solution into 10 mu L of rare earth coordination polymer probe suspension, fixing the total volume to be 100 mu L, carrying out ultrasonic treatment for 20 minutes, and detecting the fluorescence intensity of the solution at the excitation wavelength of 252nm at room temperature; and establishing a standard curve by taking the fluorescence intensity as a vertical coordinate y and taking the concentration of guanylic acid in the ham aqueous solution as a horizontal coordinate x, and drawing up a linear equation.
The rare earth coordination polymer probe is applied to the detection of the guanylic acid content in milk powder or ham.
Compared with the prior art, the invention has the beneficial effects that the organic ligand used by the rare earth coordination polymer probe prepared by the invention is nontoxic and is easily soluble in water. The prepared rare earth coordination polymer probe can be well dispersed in a water phase buffer solution and is more suitable for detecting biological samples; the rare earth coordination polymer probe is prepared by self-assembly reaction at room temperature, high temperature and longer reaction time are not needed, and the preparation process is simpler; the prepared rare earth coordination polymer probe has stable optical property, strong identifying capability on guanylic acid, quick response, response range of 0.15-20 mu M and detection limit of 0.10 mu M. Therefore, the rare earth coordination polymer probe can be used for quantitatively detecting guanylic acid in infant formula milk powder, ham and the like which are difficult to detect in the traditional mode.
Drawings
FIG. 1 is a Scanning Electron Micrograph (SEM) of terbium complex polymer probes before (a) and after (b) addition of guanylic acid.
FIG. 2 is a graph showing the linear relationship between the terbium coordination polymer probe of the present invention and the detection of guanylic acid in an aqueous solution.
FIG. 3 is a linear relationship diagram of the terbium-coordinated polymer probe of the present invention for the detection of guanylic acid in milk powder.
FIG. 4 is a graph of the linear relationship of terbium-coordinated polymer probes of the present invention for the detection of guanylic acid in ham.
FIG. 5 is a graph showing the X-ray energy spectrum analysis of terbium complex polymer probes before (a) and after (b) addition of guanylic acid.
FIG. 6 is an X-ray diffraction pattern of a terbium complex polymer probe before (a) and after (b) addition of guanylic acid.
FIG. 7 is an infrared spectrum of citrate (a), terbium complex polymer probe (b), terbium complex polymer probe (c) after addition of guanylic acid, and guanylic acid (d).
FIG. 8 is an emission spectrum of Cit/Tb before (a) and after (b) addition of guanylic acid at an excitation wavelength of 252nm, and the inset is a corresponding photograph under an ultraviolet lamp.
FIG. 9 is Tb3+: Cit/Tb-GMP fluorescence spectra at different molar ratios of Cit.
FIG. 10 is a graph of the fluorescence lifetime of Cit/Tb and Cit/Tb-GMP.
FIG. 11 shows the UV absorbance pattern of citrate (a), rare earth coordination polymer probe (b), guanylic acid (c), Tb-GMP (d), and guanylic acid added to Cit/Tb.
FIG. 12 is the effect of pH on the fluorescence intensity of Cit/Tb after addition of guanylic acid.
FIG. 13 is the change in fluorescence intensity over ten days for Cit/Tb.
FIG. 14 is a graph of the fluorescence spectrum of a terbium-coordinated polymer probe of the present invention after addition of different concentrations of guanylic acid; the concentrations of guanylic acid added from bottom to top are respectively: 0.15, 0.5, 1.5, 2.5, 3.5, 5.0, 6, 8, 10,12, 15, 18, 20 μ M, the solution system was 100mM HEPES (pH 7.4) in water, the abscissa was the wavelength and the ordinate was the fluorescence intensity (the inset is the linear relationship between the fluorescence intensity of the coordination polymer probe and guanylic acid).
FIG. 15 is the selectivity of the rare earth coordination polymer probe of the present invention for GMP; the abscissa is the interfering substance and the ordinate is the fluorescence intensity.
FIG. 16 shows fluorescence spectra of Tb-GMP, Tb-AMP, Tb-CMP, Tb-UMP and Tb-IMP.
FIG. 17 is a fluorescence spectrum of ham sample and milk powder sample in fluorescence mode (b, d) and time-resolved mode (a, c), respectively.
FIG. 18 is a schematic diagram of determination of guanylic acid by terbium complex polymer probe.
Detailed Description
The preparation method of the rare earth coordination polymer probe for detecting guanylic acid comprises the following steps of mixing a citrate solution and a rare earth metal salt solution, wherein the molar ratio of the citrate to the rare earth metal salt is (0.5-5): 5, stirring and reacting for 2-6h at room temperature, centrifuging and collecting white precipitate, wherein the rotating speed of centrifugal separation is 6000-8000 rpm, washing the collected white precipitate with deionized water, and then drying at the drying temperature of 60-80 ℃ to obtain the rare earth coordination polymer probe for detecting guanylic acid.
The rare earth metal ion in the rare earth metal salt solution is Tb3+、Eu3+At least one of (1).
The citrate is at least one of sodium citrate and potassium citrate.
The rare earth coordination polymer probe is prepared by the preparation method of the rare earth coordination polymer probe for detecting guanylic acid.
Citrate (Cit) is a derivative of citric acid present in citrus fruits; citrate is an essential intermediate in the metabolism of the citric acid cycle for all aerobic organisms. In addition, it is a water-soluble tricarboxylic acid which can provide the O atom of its carboxylic acid group with Tb3+And (4) coordination.
The chemical structure of citrate is as follows:
the chemical structure of guanylic acid (GMP) is as follows:
the rare earth coordination polymer probe is a nano particle, and the guanylic acid (GMP) can coordinate with the rare earth ions and transfer the excitation energy to the rare earth ions to sensitize Tb3+I.e. "antenna effect". However, the fluorescence is quenched by O-H vibration of the water molecules coordinated to the complex surface. Thus, utilizing a single Tb3+When guanylic acid is detected, the fluorescence is very weak, and the detection sensitivity is very low. And Cit/Tb is used as a fluorescent probe to detect guanylic acid, and the carboxyl of citrate, the phosphate group in guanylic acid and the N end are coordinated with rare earth ions, so that the coordination of the coordination between water molecules and the rare earth ions disappears, and the energy transfer between ligands and the rare earth ions is greatly sensitized.
Therefore, the invention provides a rare earth coordination polymer probe, and the citrate hardly sensitizes Tb due to almost no absorption of the citrate in the ultraviolet region3+And the rare earth coordination polymer probe emits light, so the fluorescence of the rare earth coordination polymer probe is weak. The base and phosphate groups of guanylic acid are easy to react with rare earth Tb3+Coordination, forming coordination polymer rare earth coordination polymer probe-guanylic acid with the rare earth coordination polymer probe, in the coordination polymer, because the rare earth coordination polymer probe provides effective hydrophobic environment for guanylic acid, the fluorescence intensity is greatly increased after the guanylic acid is added. Therefore, guanylic acid can be detected by a fluorescence spectrophotometry method.
The rare earth coordination polymer probe hardly emits light in an aqueous solution due to an energy mismatch between citrate and guanylate. However, after addition of guanylic acid, the coordination of water molecules and the passage from guanylic acid to Tb was eliminated3+By the conversion of fluorescence resonance energy, we observed a 15-fold increase in fluorescence. The schematic diagram of the response after addition of guanylic acid is shown in FIG. 18.
Example 1:
1. preparation of fluorescent probe of terbium coordination polymer
The preparation steps of the terbium coordination polymer probe are as follows: 1mL (100mM) of aqueous terbium nitrate solution was added dropwise to 0.5mL (100mM) of trisodium citrate solution, and stirred at room temperature for 3.5h, resulting in white precipitate. After the reaction is finished, centrifugal separation is carried out (8000rmp multiplied by 10min), white precipitate is collected, the obtained precipitate is washed by deionized water, centrifugal separation and washing by the deionized water are repeated for three times, finally, the obtained product is dried in an oven at 70 ℃ to obtain the terbium coordination polymer probe (Cit/Tb) for detecting the guanylic acid, a scanning electron microscope picture of the terbium coordination polymer probe is shown in figure 1a, and as can be seen from figure 1a, the prepared polymer probe is a nanoparticle.
The obtained terbium complex polymer probe (Cit/Tb) was dispersed in 1mL of HEPES buffer (100mM, ph7.4) to form a terbium complex polymer probe suspension for subsequent detection.
Preparation of HEPES buffer: HEPES was dissolved in pure water to prepare a HEPES buffer solution (concentration: 100mM) having a pH of 7.0, and then the pH was adjusted with a 5M sodium hydroxide solution.
The rare earth coordination polymer probe has good water dispersion performance and can be detected in a water phase.
2. Establishment of Standard Curve for detection of guanylic acid in aqueous solution
Preparing a guanylic acid standard solution (10mM), adding different volumes of the guanylic acid standard solution into 10 mu L of terbium coordination polymer probe suspension to enable the concentration of the guanylic acid in the suspension to be changed in a gradient manner (the concentrations of the guanylic acid in the suspension are 0 mu M, 0.15 mu M, 0.5 mu M, 1.5 mu M, 2.5 mu M, 3.5 mu M, 5 mu M, 6 mu M, 8 mu M, 10 mu M, 12 mu M, 15 mu M, 18 mu M and 20 mu M respectively), fixing the total volume to be 100 mu L by adjusting the amount of HEPES buffer solution, and measuring the corresponding fluorescence intensity at an excitation wavelength of 252nm after reacting for 15 minutes at room temperature; and establishing a standard curve by taking the fluorescence intensity as an ordinate y and the concentration of guanylic acid in the suspension as an abscissa x, and drawing up a linear equation as shown in figure 2.
3. Establishment of standard curve for detecting guanylic acid in milk powder
Preparing a guanylic acid standard solution (10mM), dissolving milk powder in water to obtain a milk powder aqueous solution, removing proteins in the milk powder by using an ultrafiltration centrifugal tube, adding different volumes of guanylic acid standard solutions into the milk powder aqueous solution to obtain milk powder aqueous solutions containing guanylic acid with different concentrations (the concentrations of guanylic acid in the milk powder aqueous solution are respectively 9.58mg/kg, 28.74mg/kg, 47.90mg/kg, 95.81mg/kg, 191.62mg/kg, 287.43mg/kg, 479.05mg/kg, 766.48mg/kg and 958.09mg/kg), adding 5 mu L of the milk powder aqueous solution with different concentrations and 85 mu L of HEPES buffer solution into 10 mu L of terbium coordination polymer probe suspension, fixing the total volume to 100 mu L, and detecting the fluorescence intensity of the solution at 252nm excitation wavelength at room temperature after carrying out ultrasound for 20 minutes; and establishing a standard curve by taking the relative fluorescence intensity as a vertical coordinate y and taking the concentration of guanylic acid in the milk powder aqueous solution as a horizontal coordinate x, and drawing up a linear equation as shown in figure 3.
4. Establishment of standard curve for detecting guanylic acid in ham
Preparing a guanylic acid standard solution (10mM), adding 4g of crushed ham into 2mL of deionized water, carrying out ultrasonic treatment for 1 hour, and centrifuging to remove impurities to obtain a ham water solution; adding guanylic acid standard solutions with different volumes into the ham aqueous solution to obtain the ham aqueous solution containing guanylic acid with different concentrations (the concentrations of guanylic acid in the ham aqueous solution are respectively 12.47mg/kg, 49.87mg/kg, 174.56mg/kg, 249.35mg/kg, 374.03mg/kg, 498.70mg/kg, 623.375mg/kg, 748.05mg/kg, 872.73mg/kg and 997.40 mg/kg); adding 5 mu L of ham aqueous solution with guanylic acid with different concentrations and 85 mu L of HEPES buffer solution into 10 mu L of terbium coordination polymer probe suspension, fixing the total volume to be 100 mu L, carrying out ultrasonic treatment for 20 minutes, and detecting the fluorescence intensity of the solution at the excitation wavelength of 252nm at room temperature; and establishing a standard curve by taking the relative fluorescence intensity as a vertical coordinate y and taking the concentration of guanylic acid in the ham water solution as a horizontal coordinate x, and drawing up a linear equation as shown in figure 4.
Relative fluorescence intensity refers to the fluorescence intensity in the presence of guanylic acid minus the fluorescence intensity of Cit/Tb itself.
5. Characterization of terbium coordination polymer probes and their response to guanylic acid
By passingThe chemical composition of the terbium-coordinated polymer probe (Cit/Tb) and the probe after addition of guanylic acid was analyzed by X-ray spectroscopy, as shown in FIG. 5. C. O, Tb these peaks demonstrate Tb3+And citric acid are involved in the construction of Cit/Tb, indicating that Cit/Tb is successfully synthesized, as shown in FIG. 5 a. After addition of guanylic acid, the new peak of P in the X-ray spectrum further confirms that guanylic acid is involved in the coordination with Cit/Tb, as shown in FIG. 5 b.
X-ray diffraction (XRD) analysis confirmed that the terbium coordination polymer probe (Cit/Tb) was amorphous as shown in FIG. 6 a. After addition of guanylic acid, except for Cit and guanylic acid and Tb3+The increase in the probe particle size by coordination of (a) does not observe obvious morphological change and crystal form change, as shown in fig. 1b and 6b, indicating that guanylic acid does not affect the morphology and crystal phase of terbium coordination polymer probe.
Verification of Cit and GMP with Tb by FT-IR analysis3+Binding behavior between, as shown in FIG. 7, 1589cm for pure Cit-1And 1419cm-1Has a peak value of COO-Asymmetric and symmetric telescopic vibrations. When with Tb3+When combined, these peaks were shifted to 1580cm each-1And 1408cm-1This means that Cit is bound to Tb via carboxyl groups3+And (4) coordination. Pure GMP at 978cm-1(P-O)，1627cm-1(N1-C2) and 1673cm-1(C ═ O) has an IR peak. After GMP addition to the terbium complex polymer probe, the three peaks shifted to 1000cm each-1,1639cm-1And 1679cm-1These changes indicate GMP and Tb3+The coordination occurs through P-O in GMP, N1-C2, C ═ O.
6. Fluorescence behavior of Terbium coordination Polymer probes
FIG. 8 shows the time-resolved luminescence behavior of a terbium-coordinated polymer probe. Terbium complex polymer probes exhibit very weak emission in aqueous solution due to fluorescence quenching caused by OH vibration in the coordinated water molecules. However, after addition of guanylic acid, Cit/Tb-GMP is formed, an approximately 15-fold increase in emission is observed, and the fluorescence intensity of Cit/Tb-GMP is related to the molar ratio of Cit to Tb. In the probe, the molar ratio is 5: 2.5 (Tb: Cit) produced the strongest fluorescence enhancement, as shown in FIG. 9.We attributed this fluorescence enhancement to the energy conversion of guanylic acid to Cit/Tb from GMP coordination with Cit/Tb, which resulted in a hydrophobic environment inside the terbium coordination polymer probe, minimizing the radiationless transition caused by OH vibration. The luminescence lifetime increased from 1.28ms to 1.60ms as shown in FIG. 10 after addition of guanylic acid, further demonstrating the replacement of coordinated water molecules by guanylic acid and the reduction of the excited state Tb3+Is not radiatively decayed. The long lifetime of Cit/Tb (in milliseconds) makes it suitable for complex practical sample analysis.
To explore the fluorescence enhancement mechanism, we also performed uv absorption. As shown in FIG. 11, there is a characteristic absorption of guanylic acid at a wavelength of 254 nm. When guanylic acid is added into a terbium coordination polymer probe to form Cit/Tb-GMP, the position of the maximum absorption peak at 254nm is blue-shifted to 252nm, and the ultraviolet absorbance is also obviously increased. These results indicate that, in the outer range, GMP coordination to Cit/Tb results in guanylic acid to Tb3+Excited state5D4More efficient energy transfer therebetween. Furthermore, the luminescent response of Cit/Tb to GMP is pH dependent, reaching a maximum at pH7.4, as shown in figure 12. Under other pH conditions, the significant decrease in Cit/Tb-GMP fluorescence intensity may be due to protonation of GMP in acidic medium and formation of terbium hydroxide in alkaline medium, which destroys the structure of Cit/Tb-GMP. Thus, during the experiment, HEPES buffer (100mM, pH7.4) was selected for guanylic acid determination. Within two weeks, no significant change in Cit/Tb fluorescence was observed at room temperature, indicating that our synthetic Cit/Tb was highly stable for analysis of real samples, as shown in FIG. 13.
7. Detection performance of guanylic acid in aqueous solution
To evaluate the GMP sensing performance of Cit/Tb, we investigated the addition of different GMP concentrations to the probe solution, corresponding to Tb3+ 5D4→7F5The trend of luminescence at 545nm is shown in FIG. 14. As can be seen from FIG. 14, Cit/Tb also increased in fluorescence intensity with increasing GMP concentration. The fluorescence intensity at Tb-Cit 545nm has a good linear response to guanylic acid concentration in the concentration range of 0.15-20. mu.M. Based on 3: 1 signal to noise ratioThe detection limit was 100nM, as shown in FIG. 2. Compared with other reported guanylic acid sensors, the Detection Limit (DL) of the guanylic acid sensor is 0.16 mu M, 0.27 mu M, 0.5 mu M, 5 mu M, 1.3 mu M and 8.01 mu M respectively, and the current synthesized Cit/Tb probe has more advantages for detecting GMP.
Selectivity of Cit/Tb for GMP detection
To evaluate the selectivity of the synthesized time-resolved Cit/Tb of the present application for guanylic acid, the effect of other nucleotides (CMP, UMP, AMP, IMP) was also investigated. As shown in FIG. 15, under the same conditions, these other nucleotides did not elicit significant enhancement of Cit/Tb fluorescence, probably their low affinity and binding to Tb3+The excited states are not energy matched. Fig. 16 demonstrates this conclusion. Furthermore, in view of the practical application of Cit/Tb in infant formulas and ham, we examined other ingredients including ascorbic acid (AA, commonly found in ham) and metal ions (K) commonly found in infant formulas+，Cl-，Zn2+，Mn2+，Fe2+，Cu2+) Influence on Cit/Tb fluorescence intensity. As can be seen in fig. 15, these substances do not interfere with GMP detection. These results indicate that under the current conditions, Cit/Tb is highly selective for GMP detection and that the Cit/Tb based assays prepared by us can be used for the detection of guanylic acid in infant formulas and ham.
9. Time-resolved fluorescence-based detection of GMP in infant formula and ham
Due to the long fluorescence lifetime of the probe, the background fluorescence of infant formula and ham was completely eliminated in the time-resolved mode, as shown in fig. 17. According to the results, further Cit/Tb was used to detect guanylic acid in infant formula and ham. As shown in FIGS. 3 and 4, the fluorescence intensity of Cit/Tb at 545nm showed a good linear correlation with guanylic acid concentrations in the range of 9.58-958.09mg/kg in infant formula and 9.58-958.09mg/kg in ham. Accordingly, as shown in tables 1-2, excellent recovery rates were achieved when guanylic acid was measured in infant formulas and ham. According to the relevant limit criteria, Cit/Tb currently prepared is sufficient to assess whether guanylic acid is added in excess in infant formulas and ham.
Table 1 shows the detection results of Cit/Tb prepared in example 1 on guanylic acid in milk powder
| Sample (I) | GMP concentration added (mg/kg) | Guanylic acid detected (mg/kg)a | Recovery (%) |
| 1 | 0 | 10.34 | — |
| 2 | 47.90 | 56.70±2.92 | 96.78 |
| 3 | 479.05 | 460.39±13.32 | 93.95 |
| 4 | 958.09 | 981.91±4.79 | 101.41 |
Table 2 shows the detection results of Cit/Tb prepared in example 1 on guanylic acid in ham
| Sample (I) | GMP concentration added (mg/kg) | Detected (mg/kg)a | Recovery (%) |
| 1 | 61.08 | 56.90±7.52 | 93.16 |
| 2 | 101.80 | 101.02±16.97 | 99.23 |
| 3 | 407.20 | 423.34±24.21 | 103.96 |
| 4 | 773.68 | 817.12±36.02 | 105.61 |
Example 2:
the preparation steps of the europium coordination polymer probe are as follows: 1mL (100mM) of an aqueous europium nitrate solution was added dropwise to 0.1mL (100mM) of a tripotassium citrate solution, and stirred at room temperature for 2 hours to produce a white precipitate. After the reaction is finished, carrying out centrifugal separation (6000rmp multiplied by 10min), collecting white precipitate, washing the obtained precipitate with deionized water, carrying out centrifugal separation, repeating the washing with the deionized water for three times, and finally drying the obtained product in a drying oven at 60 ℃ to obtain the europium coordination polymer probe for detecting the guanylic acid.
Example 3:
the preparation steps of the europium coordination polymer probe are as follows: 1mL (100mM) of an aqueous europium nitrate solution was added dropwise to 1mL (100mM) of a tripotassium citrate solution, and stirred at room temperature for 6 hours to produce a white precipitate. After the reaction is finished, carrying out centrifugal separation (8000rmp multiplied by 10min), collecting white precipitate, washing the obtained precipitate with deionized water, carrying out centrifugal separation, repeating the washing with the deionized water for three times, and finally drying the obtained product in an oven at 80 ℃ to obtain the europium coordination polymer probe for detecting the guanylic acid.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the overall concept of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (7)
1. The preparation method of the rare earth coordination polymer probe for detecting guanylic acid is characterized in that: mixing the citrate solution and the rare earth metal salt solution, stirring at room temperature, performing full reaction, centrifuging, collecting white precipitate, and drying to obtain the rare earth coordination polymer probe for detecting guanylic acid; the rare earth metal ion in the rare earth metal salt solution is Tb3+、Eu3+At least one of citrate and rare earth metal salt in a molar ratio of (0.5-5): 5.
2. the method for preparing a rare earth coordination polymer probe for detecting guanylic acid according to claim 1, wherein the probe comprises: stirring and reacting for 2-6h at room temperature, centrifuging at 6000-8000 rpm, washing the collected white precipitate with deionized water, and drying at 60-80 deg.C.
3. The method for preparing a rare earth coordination polymer probe for detecting guanylic acid according to claim 1, wherein the probe comprises: the citrate is at least one of sodium citrate and potassium citrate.
4. A rare earth coordination polymer probe prepared by the method for preparing a rare earth coordination polymer probe for detecting guanylic acid according to any one of claims 1 to 3.
5. The method for detecting guanylic acid using the rare earth coordination polymer probe according to claim 4, wherein: the method comprises the following specific steps: dissolving the rare earth coordination polymer probe in a water phase buffer solution to prepare a rare earth coordination polymer probe solution, adding the rare earth coordination polymer probe solution into the liquid to be detected, reacting for a certain time, detecting the fluorescence spectrum of the probe, and substituting the measured fluorescence intensity into a standard curve to obtain the concentration of guanylic acid in the liquid to be detected.
6. The method for detecting guanylic acid according to claim 5, wherein: the standard curve is established as follows:
a: establishment of a standard curve for the detection of guanylic acid in aqueous solution: preparing guanylic acid standard solutions, adding guanylic acid standard solutions with different volumes into 10 mu L of rare earth coordination polymer probe suspension to enable the concentration of guanylic acid in the suspension to be changed in a gradient manner, fixing the total volume to be 100 mu L by adjusting the amount of HEPES buffer solution, reacting for 15 minutes at room temperature, and measuring the corresponding fluorescence intensity of the guanylic acid standard solutions at the excitation wavelength of 252 nm; establishing a standard curve by taking the fluorescence intensity as a vertical coordinate y and the concentration of guanylic acid in the suspension as a horizontal coordinate x, and drawing up a linear equation;
b: establishment of a standard curve for detecting guanylic acid in milk powder: preparing a guanylic acid standard solution, dissolving milk powder in water to obtain a milk powder aqueous solution, removing proteins in the milk powder by using an ultrafiltration centrifugal tube, adding different volumes of guanylic acid standard solutions into the milk powder aqueous solution to obtain milk powder aqueous solutions containing guanylic acid with different concentrations, adding 5 mu L of the milk powder aqueous solutions containing guanylic acid with different concentrations and 85 mu L of HEPES buffer solution into 10 mu L of rare earth coordination polymer probe suspension, fixing the total volume to 100 mu L, carrying out ultrasonic treatment for 20 minutes, and detecting the fluorescence intensity of the solution at 252nm excitation wavelength at room temperature; establishing a standard curve by taking the fluorescence intensity as a vertical coordinate y and taking the concentration of guanylic acid in the milk powder aqueous solution as a horizontal coordinate x, and drawing up a linear equation;
c: establishment of a standard curve for detection of guanylic acid in ham: preparing a guanylic acid standard solution, adding 4g of crushed ham into 2mL of deionized water, carrying out ultrasonic treatment for 1 hour, and centrifuging to remove impurities to obtain a ham aqueous solution; adding different volumes of guanylic acid standard solutions into the ham aqueous solution to obtain ham aqueous solutions containing guanylic acid with different concentrations; adding 5 mu L of ham aqueous solution with different concentrations of guanylic acid and 85 mu L of HEPES buffer solution into 10 mu L of rare earth coordination polymer probe suspension, fixing the total volume to be 100 mu L, carrying out ultrasonic treatment for 20 minutes, and detecting the fluorescence intensity of the solution at the excitation wavelength of 252nm at room temperature; and establishing a standard curve by taking the fluorescence intensity as a vertical coordinate y and taking the concentration of guanylic acid in the ham aqueous solution as a horizontal coordinate x, and drawing up a linear equation.
7. The use of the rare earth coordination polymer probe of claim 4 in detecting guanylic acid content in milk powder or ham.
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